Mrs1220

Foetal calf serum (FCS) was obtained from PAA Laboratories (Wokingham, UK). Furimazine was purchased from Promega (Southampton, UK). Bicinchoninic acid protein assay kit and white 96-well microplates were obtained from Thermo Fisher Scientific (Waltham, MA, USA). GF/B filter plates and Microscint-O were from PerkinElmer (Groningen, The Netherlands). CA200645 was obtained from HelloBio (Bristol, UK). The synthesis of AV039 was described in Vernall et al. as compound 19 [34 (link)], while the synthesis of XAC-S-ser-S-tyr-X-BY630 (compound 27) and XAC-S-ser-S-tyr-X-BYFL (compound 28) was described in Vernall et al. 2013 [33 (link)]. PSB-11 and MRS1220 were purchased from Tocris Bioscience (Bristol, UK), and NECA was obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). [³H]8-Ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]-purin-5-one ([³H]PSB-11) was kindly donated by Prof. C.E. Müller (University of Bonn, Germany) and its synthesis described in Müller et al. [35 (link)]. 1-Benzyl-8-methoxy-1H,3H-pyrido[2,1-f]purine-2,4-dione (compound 5) synthesis was described in Priego et al. as compound number 3 [36 (link)] and referred in Xia et al. [37 (link)] as compound number 5, while LUF7565 synthesis was described in Xia et al. as compound 27 [30 (link)]. All other chemicals and reagents were obtained from Sigma-Aldrich (Gillingham, UK).

MRS 1220 was obtained from Tocris Cookson (Avonmounth, UK). CA200645 and ABEA-X-BY630 were obtained from CellAura Technologies (Nottingham, UK). VUF 5455 was synthesized by B. Kellam (Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham, UK). Fetal calf serum was obtained from PAA Laboratories (Yeovil, UK). All other reagents, including xanthine amine congener (XAC), were obtained from Sigma-Aldrich Inc. (Poole, UK).

All chemical reagents were purchased from
Sigma-Aldrich. The aCSF buffer was composed of 126 mM NaCl, 2.5 mM
KCl, 1.2 mM NaH₂PO₄, 2.4 mM CaCl₂·2H₂O, 1.2 mM MgCl₂·6H₂O, 25 mM NaHCO₃, 11 mM glucose, and 15 mM tris(hydroxymethyl)
aminomethane dissolved in deionized water (Milli-Q Biocel; Millipore,
USA). The aCSF was freshly prepared before the experiments and adjusted
to pH 7.4 for brain slice experiments. Adenosine and inosine were
purchased from Acros Organics (Morris Plains, NJ, USA). Stock solutions
(10 mM) were prepared in 0.1 M perchloric acid and diluted to a 1
mM concentration with aCSF.
DPCPX (A₁ Adenosine receptor
antagonist) and MRS 1220 (A₃ Adenosine receptor antagonist)
were purchased from Tocris Bioscience (Minneapolis, USA). A 10 μM
amount of DPCPX and 5 μM of MRS 1220 were freshly prepared by
dissolving in 1 mL of dimethyl sulfoxide (DMSO) through sonication
and diluted with an aCSF buffer.

MRS1220 (Tocris, Bristol, UK) was used as a selective antagonist of A3AR. MRS1754 (Tocris) was used as a selective antagonist of A2BAR. Doxorubicin (Laboratorio de Chile, Santiago, Chile) and teniposide (Tocris) were used as drug substrates of MRP3.

Female C57BL/6 (B6) and CD73^-/- mice were purchased from Nanjing University Animal Institute (China) and housed and maintained in the animal facilities of Tianjin Medical University. Institutional approval was obtained, and institutional guidelines regarding animal experimentation were followed. The mice used in the experiments were all 12- to 14-week-old females. The ARA1 antagonist DPCPX, the ARA2A antagonist SCH58261, the ARA2B antagonist MRS1754, the ARA3 antagonist MRS1220, and 5′-AMP were purchased from Tocris (R&D, United States). Primary antibodies against mouse CD73 and Na⁺/K⁺-ATPase were purchased from Santa Cruz (United States). PE-, FITC- or APC-labeled antibodies against mouse GFAP, CD73, CD45, CD24, and GLAST and the isotope control of the anti-CD73 antibody were purchased from eBioscience (United States).